Detection of Semi-polar Vitamins and Nutrients in ...

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Honors Research Projects The Dr. Gary B. and Pamela S. Williams HonorsCollege

Spring 2018

Detection of Semi-polar Vitamins and Nutrients inBiological SamplesJennifer [email protected]

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Recommended CitationJanovick, Jennifer, "Detection of Semi-polar Vitamins and Nutrients in Biological Samples" (2018). HonorsResearch Projects. 741.http://ideaexchange.uakron.edu/honors_research_projects/741

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Detection of Semi-polar Vitamins and Nutrients in Biological Samples

Jennifer Janovick

Department of Chemistry

Honors Research Project

Submitted to

The Honors College

2

Abstract

Metabolomics is used to examine metabolite fluctuations in biological systems and enables

the diagnosis of metabolic disorders and the identification of new therapeutic targets.1 Liquid

chromatography-mass spectrometry (LC-MS) was utilized to develop a new method using a T3

column to separate small molecule nutrients and vitamins in a tissue sample. Initial tests of

column performance used standard solutions; the amount of metabolites identified and the

elution profile of these metabolites was examined. Both the standard solution and sample of

brain tissue were tested with the final method developed. Six metabolites were identified in the

standard and 31 were identified in the tissue sample. Two of these metabolites were the same:

creatine and pyridoxine. However, broad peaks for some metabolites were observed in the

chromatography. The method developed was able to separate and identify metabolites from a

biological sample, however, based on the multiple elution points of certain compounds during

both the standard and the tissue sample, it was determined that the method should be further

modified.

Introduction

Metabolomics uses a systematic approach to examine alteration in small molecular

metabolites produced within an organism or cell.2 An organism’s metabolism involves a number

of complex reactions that work together to maintain homeostasis. Small molecule vitamins are

cofactors that facilitate these reactions and are used in processes such as protection against

environmental pathogens and the removal of free radicals.3 Additionally, deficiencies in vitamin

E and C can lead to neurological lesions and scurvy, respectively.4 Biotin, commonly known as

vitamin B7, is utilized in pyruvate carboxylase which converts pyruvate to oxaloacetate by using

3

a swinging arm to transport carbon dioxide. The oxaloacetate produced can then be used to fuel

the tricarboxylic acid cycle (TCA) for aerobic respiration or oxaloacetate can enter

gluconeogenesis if an organism needs to produce glucose.2 Additionally, vitamin B1 or thiamine,

is involved in the production of many cofactors such as thiamine diphosphate (TPP) which is

used in pyruvate dehydrogenase to convert pyruvate to acetyl CoA.5 The production of ATP is

crucial to the survival of an organism. Metabolites are often analyzed by examining biofluid or

tissue samples. By examining these, one can gain a better understanding of the fluctuations in

metabolism which can be useful in many fields including medicine and drug development.6 It is

for these reasons, among others, why it is necessary to accurately quantify vitamin levels within

biological systems.

Liquid chromatography is a method of separating molecules in a mixture based on the

interactions between analytes and two phases: a mobile phase and a stationary phase.7 In modern

liquid chromatography, stationary phases are contained within the analytical column and

composed of beads. Often, the beads are composed of silica and the true stationary phase is a

layer of liquid that surrounds the bead which allows for chemical interactions with the mobile

phase.7 The mobile phase in this project is comprised of mixtures of polar and nonpolar solvents.

The polar solvent facilitates interactions with the polar components of the sample and elution

from the column while the nonpolar component allows the nonpolar components in the sample to

be eluted.8 In order to maximize the separation of compounds with diverse chemical properties,

essential for the comprehensive detection of endogenous metabolites, solvents gradients are

performed prior to detection by mass spectrometry. Depending on the chemical composition of

the stationary phase, there a number of interactions between the analyte and stationary phase of

the column that promote analyte retention; some of these interactions can be seen in Figure 1.

4

Figure 1. Displays the general chemical interactions between analytes and the stationary phase in column

chromatography.

Reversed-phase liquid chromatography (RPLC) usually utilizes a nonpolar stationary phase

often composed of hydrocarbon chains attached to silica beads and a polar mobile phase. In

contrast, normal-phase liquid chromatography uses a polar stationary phase and a nonpolar

mobile phase, although this method is less common.10 RPLC is designed to allow the most polar

components to elute first, while nonpolar components are retained. The mobile phase can be

considered an isocratic elution or gradient elution; the mobile phase is held constant in an

isocratic elution and there are fluctuations in the mobile phase in a gradient elution.7 The T3

column utilized was composed of a stationary phase containing C18 chains attached to silica

beads, thus classifying this experiment as RPLC, and allowing for a 100% aqueous mobile

phase.

Liquid chromatography is often coupled with mass spectrometry in order to separate and

quantify biological samples, such as small molecular vitamins and nutrients. Mass spectrometry

is used to identify samples using a mass-to-charge ratio. A mass spectrometer contains an inlet,

ion source, and analyzer. The inlet introduces the sample to an ion source where it bombards the

sample with electrons to fragment the compounds into different and distinct ions.10 The ions

Hydrophilic

Interactions

Electrostatic

Interactions

Nonpolar

Interactions

5

produced are separated based on their mass-to-charge ratio with a mass analyzer. An ion

transducer is used to convert the values associated with the mass-to-charge ratios into an

electrical signal which can then be detected.7 The mass spectrometer utilized in this project uses

a number of quadrupoles. Following chromatographic separation and ionization, a sample is

introduced into a quadrupole, Q0, where the ions are aligned to be introduced into the next

quadrupole, Q1. Q1 separates the ions by charge before they are introduced into Q2 where they

are fragmented into distinct pieces. The ion fragments enter a time of flight chamber where the

lower masses travel through faster, while those that are larger take more time and thus travel

slower.

The goal of this project was to accurately detect and quantify small molecule vitamins and

nutrients by developing a new chromatography method and compare it with a previously

established method. By performing this experiment a better understanding of the analytical

measurements of vitamins in biological samples was obtained. Additionally, the techniques and

principles of chromatography and mass spectrometry, both of which are commonly used in

chemical research, were practiced. Liquid chromatography-mass spectrometry (LC-MS)-based

metabolic profiling is a common method to analyze vitamin levels and examine their role in

cellular function.11 This method allows the simultaneous measurement of thousands of small

molecule vitamins and nutrients in cells or biofluids. Additionally, this could give information on

metabolic changes associated with disease as well as how potential therapeutic effects of

vitamins.12 This project developed a method for detection of small molecule nutrients with the

Waters T3 column. Standard solutions were used to test each method and determine which

components could be detected before a biological sample was tested.

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Materials and Methods

Chemicals

All chemicals were purchased from Sigma Aldrich (St. Louis, MO). The standard solution

was prepared by dissolving the following compounds in 20 mL of water, methanol, or

chloroform to a final concentration of 1 mM; L-aspartic acid, nicotinamide, 1,2-dipalmitoyl-pc,

L-cysteine, pyruvic acid, tryptophan, biotin, L-arginine, alpha-lipoic acid, pyridoxine, creatine,

levodopa, nicotinic acid, L-ascorbic acid, glutathione, L-cystine, folic acid, hydrocortisone, and

thiamine. The structure for the components of the standard solution can be seen in Appendix 1.

Sample calculations for the determination of concentrations can be seen in Appendix 2.

LC-MS

A T3 column (Waters, XSelect HSS T3 5 µm 2.1x150 mm) was used for all experiments. For

each gradient a mobile phase was composed of differing concentrations of HPLC-grade water or

acetonitrile. The mass spectrometer, the TripleTOF 5600+ (SCIEX), was calibrated using the

following settings: flow rate 8 μL/minute, syringe diameter 4.610 nm, temperature 300°C,

ionspray voltage floating 5000, declustering potential 1000, % transmission (TIC) 1, collision

energy 10. All samples were run in positive mode.

Metabolite Isolation from Mouse Brain

All procedures were approved by the institutional care and use committee at the University of

Akron (protocol number 1609 19SMD). Mouse brain was isolated from a C57BL/6 mouse and

the brain was weighed. The total mass of the brain was 0.5466 grams and the brain was separated

into three sections with the following masses: 0.0612 grams, 0.0475 grams, and 0.0514 grams.

7

One hundred microliters of methanol was added to each sample and the sample was vortexed for

30 seconds. The samples were placed in liquid nitrogen for 1 minute. They were homogenized

and vortexed for 30 seconds; this process was repeated for an additional two times. The samples

were subsequently frozen in liquid nitrogen for 30 seconds then 750 µL of CHCl3 in methyl

water (1:2) was added along with 250 µL of chloroform, and 250 µL of HPLC water. The

samples were vortexed for 30 seconds and incubated at -20°C for 1 hour. The samples were

centrifuged at 5,000 rpm at 4°C for 5 minutes. The top aqueous layer and bottom organic layers

were transferred to new centrifuge tubes and labeled. Two hundred microliters of 35%

acetonitrile was added to the samples and the samples were then vortexed. One hundred

microliters of the sample was added to each vial, there were 6 vials total, which were tested.

Data Analysis

METLIN was used to compare the known fragmentation patterns of each metabolite with the

fragmentation resulting from each method. In order to designate a peak as a match, the mass

needed to be within 0.05 and 3 peaks were needed to determine a compound. Appendix 3 shows

example chromatograms of the standard solution using Method 4. Elements was used to analyze

the results of the tissue sample.

Results and Discussion

We first examined a twenty six minute gradient that started with a 100% aqueous mobile

phase (Figure 2).

8

Figure 2. Method 1 and compounds identified.

Method 1 was developed with the idea that the polar compounds would be eluted much faster

than the nonpolar compounds, therefore by using 100% water during the first few minutes, the

polar compounds would be eluted quickly while the nonpolar compounds would be retained.

Reduced glutathione was the first component of the standard solution eluted at 13.07 minutes. At

this time, there was approximately 30% water and 70% acetonitrile running through the column.

Reduced glutathione is a tripeptide composed of cysteine, glutamic acid, and glycine. Cysteine is

uncharged and polar, glutamic acid is charged and polar, and glycine is nonpolar.2 These

components of reduced glutathione indicate the molecule has many diverse areas that have the

potential to interact with the column or the mobile phase. Similar to glutathione, levodopa was

eluted at 13.30 minutes and possesses a number of polar functional groups. The hydroxyl groups

on the ring are electron donating, adding resonance and stability along with a constant source of

electrons to the ring. Tryptophan, biotin, and hydrocortisone were eluted at 21.21 minutes, 21.39

minutes, and 23.46 minutes respectively, when the mobile phase contained only acetonitrile. All

three compounds possess cyclic components; tryptophan possesses an indole, biotin contains two

9

five membered rings, while hydrocortisone possesses three six membered rings and one five

membered ring. With this method, the first compound was eluted after 13 minutes avoiding the

void volume; however, eluting all standards after 13 minutes increases the potential for

compounds to have the same retention times. Pyruvic acid was not seen; however, only positive

mode was run. Positive mode indicates only the positive fragments of the molecule are detected,

therefore, if negative mode were run, it is possible that pyruvic acid could have been detected.

We subsequently developed a second gradient method to determine if we could elute

components at an earlier time than 13 minutes.

Figure 3. Method 2 and compound retention for T3 column.

Method 2 was developed as a modification of Method 1 and a number of additional

metabolites were added to the standard solution to determine if the new method could detect

them; these results can be seen in Figure 3. Because the amount of compounds in the standard

10

increased, the method time was also increased in order to promote the elution of these

compounds and to promote proper separation between compounds. Additionally, there was more

fluctuation in the polar and nonpolar phases in order to elute some of the compounds in the

standard that were not easily eluted. Glutathione was eluted at 2.03 minutes where approximately

5% acetonitrile and 95% water was being run through the column. This is vastly different than

the previous method where glutathione was eluted with a higher percentage of acetonitrile than

water. Approximately 40% acetonitrile and 60% water was needed to elute L-cysteine at 12.3

minutes. L-tryptophan and biotin were eluted at 23.2 minutes and 24.8 minutes, respectively,

where the solvent concentrations were approximately 5% water and 95% acetonitrile, similar to

the conditions at which the compound was eluted in Method 1 which was 0% water and 100%

acetonitrile. The semi-polar characteristics of tryptophan make its retention and quantification

challenging. Similar to the time frame in Method 1, hydrocortisone was the last compound to be

eluted, however, in the second method it was eluted with approximately 5% acetonitrile and 95%

water. This inconsistency is particularly interesting because the structure of hydrocortisone

indicates the molecule is overall nonpolar in nature, therefore one would expect a higher

concentration of the nonpolar component of the mobile to allow elution. Although the number of

standards eluted did not increase with Method 2, the first component was eluted around 2

minutes instead of 13 minutes, therefore Method 2 did show some improvement compared to

Method 1.

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Figure 4. Method 3 and compound retention times.

Method 3 was developed with a similar pattern as Method 2, however the time at which the

95% water and 95% acetonitrile was held constant longer. This was done to increase the

likelihood of the highly polar and highly nonpolar components of the standard solution to move

down the column and be eluted. Additional compounds were added to the standard solution to

run the third method developed which can be seen in Figure 4. L-glutamine was eluted at 10.90

minutes where the mobile phase was approximately 25% acetonitrile and 75% water.

Structurally, L-glutamine is an overall polar amino due to the differences in electronegativity

between nitrogen and oxygen. Alpha-lipoic acid was eluted at 11.30 minutes where the mobile

phase was approximately 30% acetonitrile and 70% water indicating the polarity of the

compound. Levodopa, nicotinamide, and pyridoxine were eluted at 12.50 minutes, 12.50

minutes, and 12.80 minutes, respectively. Levodopa and pyridoxine both possess hydroxyl

groups; this adds polar functional groups to the structure, however the carbonyl on levodopa and

the placement of the hydroxyl groups on pyridoxine balance some of the polar groups to yield

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nonpolar qualities. Similarly, nicotinamide contains a carboxyl group and a six membered ring

with a nitrogen attached to it; the ring provides stability and a nonpolar component while the

carboxyl group provides polarity. Nicotinic acid is structurally similar to nicotinamide because

both compounds have a six member ring with a nitrogen, however, nicotinic acid has a

carboxylic acid group while nicotinamide has an amide. Nitrogen is less electronegative than

oxygen therefore the dipole moment would be pointed towards the oxygen whereas the dipole

moment on nicotinamide would be more distributed between the oxygens on the carboxylic acid.

This would result in nicotinic acid being slightly less polar than nicotinamide and would explain

why it was eluted at 14 minutes where there was approximately 45% acetonitrile and 55% water.

Glutathione was eluted at 16.74 minutes where the mobile phase was approximately 55%

acetonitrile and 45% water; this is more similar to the first method where the compound was

eluted with 70% acetonitrile and 30% water. This shows glutathione has both polar and nonpolar

components. L-tryptophan, biotin, and hydrocortisone were eluted at 24.96 minutes, 26.42

minutes, and 29.68 minutes, respectively. The majority of the mobile phase when each of these

compounds were eluted was acetonitrile which is consistent with the findings of the first two

methods. Method 3 seemed to be an improvement over the first two methods; ten compounds

were identified as opposed to six and there seemed to be adequate separation between compound

eluents.

13

Figure 5. Method 4, retention times, and retention factors.

Method 4 was selected for the analysis of the biological sample; however, due to time

constraints, an additional method to test another biological sample was not able to be developed.

However, because few metabolites were seen past 30 minutes in the previous two methods,

Method 4 was shortened. Additionally, because the column is nonpolar, and nonpolar

compounds are harder to elute, the percent of acetonitrile was increased to 100% in the method,

similar to Method 1. Figure 5 shows the results of the fourth method developed.

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Figure 6. Total ion chromatogram using Method 4.

Figure 6 shows the total ion chromatogram for the standard solution which indicates there

was a large amount of an unknown substance eluted within the first 5 minutes of the method;

however this was unable to be identified. Creatine was eluted between 10 and 11 minutes where

the mobile phase was approximately 40% acetonitrile and 60% water. When examining the

chromatogram, one can see that although the intensity is low, there is only one place where it

was eluted. In contrast to the first three methods where L-tryptophan was eluted with 95%-100%

acetonitrile, the compound was eluted in this method with approximately 50% water and 50%

acetonitrile around 11 minutes where most of the tryptophan was eluted, however there was a

small amount eluted around 16 minutes. Pyridoxine could be seen in two places; around 11

minutes where the mobile phase was slightly more polar than nonpolar, and around 16 minutes

where the amount of water and acetonitrile were approximately equal. However, both intensities

are relatively low when comparing to the intensity of the total ion chromatogram. The third

method eluted pyridoxine with 30% acetonitrile and 70% water suggesting the molecule is more

polar than nonpolar, however, because it was eluted at two different mobile phase

concentrations, this shows there are different polar and nonpolar components. These components

could interact with the column in different ways each time a sample is run; the interactions

15

between the sample and the column for the first method won’t necessarily be the same as the

second or third methods. Biotin was eluted at two separate points in the method; the first was

around 11 minutes with approximately 40% acetonitrile and the second was approximately 16

minutes with approximately 50% water and 50% acetonitrile. Appendix 3 shows some of the

chromatograms from the standard solution and indicates the majority of the biotin was eluted

around 16 minutes. This was similar to the mobile phase concentrations of the previous methods

used to elute the compound. Similar to pyridoxine, levodopa seemed to be eluted at a number of

times. The first elution was around 11 minutes where the mobile phase was approximately 40%

acetonitrile and 60% water while the second elution was around 21 minutes where there was

100% acetonitrile and 0% water. This is similar to what was seen in Method 1 where levodopa

was eluted with approximately 80% acetonitrile and 20% water whereas in Method 3 it was

eluted with approximately 30% acetonitrile and 70% water. However, the ion chromatogram for

levodopa shows no distinct peaks, although there is an increase in intensity around 10 minutes.

Nicotinic acid was eluted with approximately 40% acetonitrile and 60% water which was similar

to Method 3, however, the intensity of the ions were under 2000 yet there was a distinct peak.

The retention factor was calculated for each peak present in Method 4 in order to give one an

understanding of how long the components of the standard are in the stationary and mobile

phases. Generally, this value is between 1 and 5.7 However, the values calculated are much

higher than the general range thus indicating the analytes in the standard solution are located in

the stationary phase longer and they are difficult to elute. From these values it can be determined

that the method developed was not the best, therefore this further indicates the final method

should be further modified.

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Table 1. Shows the metabolites identified using Method 4 along with the average mass-to-charge ratio,

and the average time eluted for the tissue sample.

17

Figure 7. Total ion chromatogram for the tissue sample along with the deviations between the 6 samples.

Elements was used in order to determine which metabolites could be identified from the

tissue sample; these results can be seen in Table 1 and Figure 7. There were two metabolites

detected in both the standard solution and the tissue sample using Method 4: creatine and

pyridoxine. These images can be seen in Appendix 4. Although the peak for creatine in the

standard solution was not a high intensity, and the peak in the tissue sample was relatively broad,

the compound was eluted around 10 minutes in both. Similarly, pyridoxine was eluted around 11

minutes in both samples although it was eluted a second time around 16 minutes when the

standard solution was run. The chromatogram for the standard indicates there was a greater

amount of pyridoxine eluted at 16 minutes rather than 11 minutes, however the intensity of both

peaks were two of the highest after examining all the eluted compounds in the standard.

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There were a number of inconsistencies in the standard solutions regarding the time and

gradient concentrations needed to elute certain components. The majority of biotin was eluted in

each method with the mobile phase having a larger or equal part concentration of acetonitrile

compared to water (100%, 95%, 95%, and 50%, for Method 1, Method 2, Method 3, and Method

4, respectively). However the time at which it eluted differed slightly; it was eluted in the early

to mid-twenty minute mark for the first three methods, but around 11 minutes and again at 16

minutes in the last method. It is possible biotin was eluted slightly earlier in Method 4 due to the

increase in concentration; between 0 and 15 minutes the increase of acetonitrile is constant, and

at 15 minutes, the slope increased, exposing the column to more of the nonpolar component. This

sudden influx could have resulted in the early elution time. The elution of glutathione also

showed a number of inconsistencies; Method 1 and Method 3 indicate the compound was eluted

with a higher concentration of acetonitrile between 13 and 17 minutes. In contrast, Method 2

indicates shows it was eluted with approximately 5% acetonitrile at 2.03 minutes. All three

methods show glutathione was eluted while acetonitrile was increasing, however, the time

differences are unaccounted for. In the first three methods run, hydrocortisone was the last

compound eluted; this could be due to rigidity associated with the four rings in the structure.

Additionally, the molecule itself suggests it is nonpolar as a whole, although there are some polar

components. This idea is supported by the results shown in Method 1 and Method 3 where 80%

acetonitrile and 70% acetonitrile, respectively, were present at the detection of the compound.

However, in Method 2, the mobile phase was 5% acetonitrile and 95% water when

hydrocortisone was eluted at 36.5 minutes. Because the compound was eluted so late, it’s

possible the interactions between the molecule and column became weaker over time and it was

finally eluted. The elution of L-tryptophan was relatively consistent through the first three

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methods; elution occurred between 20 and 25 minutes with a concentration of acetonitrile of

either 95% or 100%. However, Method 4 showed the majority of L-tryptophan being eluted

much earlier, around 11 minutes, with only about 40% acetonitrile. The polarity associated with

the carboxylic acid part of the molecule could have caused it to be eluted in a more polar

environment compared to the other methods. Levodopa was seen in Method 1, Method 3, and

Method 4. The molecule was eluted at 13.3 minutes with approximately 80% acetonitrile and at

12.5 minutes with 35% acetonitrile in Method 1 and Method 3, respectively. Interestingly, it

appeared levodopa was eluted at 2 different points in Method 4: at 11.45 minutes and 21.35

minutes where the amount of acetonitrile was about 40% and 100% respectively. Most of the

levodopa was eluted at the 11.45 minutes; however, the nonpolar nature of the molecule could

have required the pure acetonitrile to remove it completely. Finally, pyridoxine was eluted in the

final two methods; the times at which it was eluted was relatively consistent; 12.8 minutes in

Method 3 and 11.26 minutes and 15.99 minutes in Method 4. However, the concentration of the

mobile phase was different. Method 3 required approximately 35% acetonitrile while the first

elution Method 4 used 40% acetonitrile and 50% acetonitrile for the second elution point.

These inconsistencies could be due to a number of reasons. The standard solution was remade

multiple times throughout the method development process; at times there were less components

while there were more during other methods. Human error in measuring or determining the

correct concentration could have resulted in different concentrations of each standard solution.

Although excess care was always taken, this was relatively unavoidable to an extent. Many of

the compounds used in the standard, such as aspartic acid and biotin, possess both polar and

nonpolar components. These components could have reacted slightly differently with the column

and the mobile phase in each method run; the nonpolar component sticking to the column in an

20

early method may not stick to the column in a later method run, while another part of the

molecule would. The path the solution takes down the column each run can vary as well.

Additionally, it is possible the compounds in the standard solution reacted with one another

which could have inconsistencies of in elution times.

Method 1 and Method 4 allowed for the elution and identification of many of the components

of the standard solution, however, the components that were identified were all eluted within 11

minutes of each other. Method 2 and Method 3 both had longer run times and it is likely this

resulted in the more separated elution times. Method 1, Method 2, and Method 3 seemed to elute

the majority of the compound at one time, as opposed to Method 4 where multiple elution peaks

were observed for almost all of the compounds eluted. Additionally, Method 4 produced broad

peaks with low intensities. Based on these factors, if another biological sample were available to

be run, Method 3 or a variation of Method 3 should be used.

The ability to separate and accurately quantify metabolites from one another possesses a vast

number of practical applications that can be applied to many fields. The ability to monitor the

amount of metabolites within a cell at a given time, allows one to monitor the effects of

medication or pathology associated with disease. A number of other separation methods have

been developed for LC-MS-based metabolomics, including the use of nonpolar and polar

columns. For example, a nonpolar C18 column was used to identify biomarkers in blood samples

of individuals who have esophageal adenocarcinoma in order to improve possible treatment

options. The mobile phase consisted of 0.2% acetic acid in water as the A portion and 0.2%

acetic acid in methanol as the B portion. Between 0 and 13 minutes, the B phase increased from

2% to 98% and was held at 98% for 6 minutes. The Agilent database was used in order to

identify the metabolites within the sample; over 5000 were identified, however, 40 metabolites

21

were identified to have a significant difference between healthy individuals and those who were

considered high risk.13 Similar to the method developed in this project, this method utilized a

nonpolar stationary phase consisting of carbon chains and a mobile phase consisting of polar and

nonpolar components. However, the method used to identify metabolites associated with EAC

was able to identify many more metabolites within a shorter amount of time. The total time for

the method was 19 minutes while the method used on the brain sample was 26 minutes. This

indicates the method developed in this project may have future potential if it is further modified.

Additionally, HILIC columns have shown to separate components of a standard solution and

specifically water was used as the polar solvent of the mobile phase while acetonitrile was the

nonpolar solvent. In a previous method developed, 98% acetonitrile which was held constant for

the first 30 seconds, then the percent of acetonitrile decreased and reached 95% at 1 minute. The

percent of acetonitrile continued to decrease and reached 80% at 5 minutes, 46% at 6 minutes,

14.7% at 13 minutes, and 0% at 17 minutes. From 17.1 minutes until 23 minutes, acetonitrile

was at 100%.14 Two common metabolites were found using this method and the method

established in this project: levodopa and tryptophan. The method utilizing the HILIC column

allowed for the elution of levodopa at 8.86 minutes and tryptophan at 11.34 minutes. Using the

T3 column, levodopa was eluted at 11.45 minutes and 21.349 minutes, and tryptophan was

eluted at 10.99 minutes, 11.81 minutes, and 16.16 minutes. When separating components of a

sample it is often desired that the vast majority of the compound is eluted at a single time as

opposed to multiple elution points. Therefore, from this data it can be determined that the

method developed in this project was not as effective in separating metabolites as the previous

developed method.

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Conclusion

This project utilized a T3 column in order to test a number of methods developed with the

purpose of separating metabolites in a biological sample then using mass spectrometry to

identify and quantify the metabolites. Overall, it was determined that the principle of using LC-

MS was successful in identifying and quantifying metabolites and small molecular vitamins.

Biotin, reduced glutathione, hydrocortisone, and L-tryptophan were consistently eluted

throughout the methods. The final method used to test the tissue sample was successful in

separating and identifying metabolites in a biological system. However, the low intensities and

broad peaks indicate the method could be modified in order to further improve the number of

metabolites identified and the separation between them.

23

Appendix 1: Structures

Figure 1A. Structures of analytes contained in the standard mix A) L-arginine, B) L-ascorbic

acid, C) L-aspartic acid, D) biotin, E) creatine, F) L-cysteine, G) L-cystine, H) 1,2-dipalmitoyl-

pc, I) levodopa, J) folic acid, K) glutamine, L) reduced glutathione, M) hydrocortisone, N)

nicotinic acid, O) pyridoxine, P) pyruvic acid, Q) nicotinamide, R) alpha lipoic acid, S) thiamine,

T)tryptophan. All structure were drawn in Chemdraw with the following files: L-arginine.cdx, L-

ascorbic acid.cdx, L-aspartic acid.cdx, Biotin.cdx, Creatine.cdx, L-cysteine.cdx, L-cystine.cdx,

1,2-Dipalmitoyl-pc.cdx, Folic acid.cdx, Glutamine.cdx, Reduced glutathione.cdx,

Hydrocortisone.cdx, Levodopa.cdx, Alpha lipoic acid.cdx, Nicotinamide.cdx, Nicotinic acid.cdx,

Pyridoxine.cdx, Pyruvic acid.cdx, Thiamine.cdx, Tryptophan.cdx.

24

Appendix 2: Sample Calculations

Determining the necessary mass of the needed compound for 20 mL

Mass of L-arginine = 176.124 g/1 mol

176.124 𝑔

1 𝑚𝑜𝑙×

. 001 𝑚𝑜𝑙

1 𝐿×

1 𝐿

1000 𝑚𝐿×

20 𝑚𝐿= 𝑔𝑟𝑎𝑚𝑠 𝑛𝑒𝑒𝑒𝑑𝑒𝑑

𝑔𝑟𝑎𝑚𝑠 𝑛𝑒𝑒𝑑𝑒𝑑 = .00352 𝑔𝑟𝑎𝑚𝑠 = 3.52 𝑚𝑔 𝐿 − 𝑎𝑠𝑐𝑜𝑟𝑏𝑖𝑐 𝑎𝑐𝑖𝑑

Determining the amount of each solution for the standard

Concentration of solution = 1 mM

Concentration of standard = .0005 mM

(𝑀1) × (𝑉1) = (𝑀2) × (𝑉2)

(0.0005 𝑚𝑀) × (5 𝑚𝐿) = (1𝑚𝑀) × (𝑋 𝑚𝐿)

𝑋 = 0.0025 𝑚𝐿 = 2.5 𝜇𝐿

Determining retention factor

k’ = retention factor

tR = retention time

tM = dead time

𝑘′ =𝑡𝑅 − 𝑡𝑀

𝑡𝑀

𝑘′ =10.9 − 0.1

0.1

𝑘′ = 108.0

25

Appendix 3: Method 4 Standard

Image 3A. Elution of biotin.

Image 3B. Elution of nicotinic acid.

Image 3C. Elution of pyridoxine.

26

Appendix 4: Biological Sample

Image 4A. Shows the elution of creatine using Method 4.

Figure 4B. Shows the elution of pyridoxine using Method 4.

27

Appendix 5: Safety

A number of safety considerations were accounted for during data collection. Gloves were

worn at all times while handling chemicals. None of the reagents used had high levels of toxicity,

therefore a fume hood was not necessary for making the standards. The mass spectrometer is a

circuit therefore care was taken in order to avoid electrocution. A biological hood was used to

perform the Bligh Dyer extraction in order to minimize the possibility of contamination.

Additionally, gloves were worn while handling all biological samples. The GHS classification

was determined for all compounds used to make the standard solutions; these can be seen in

Table 5A.

Compound GHS Classification

L-arginine Warning

L-ascorbic acid None

L-aspartic acid None

Biotin None

L-cysteine None

L-cystine None

1,2-dipalmitoyl-pc None

Folic acid Warning

L-glutamine None

Glutathione (reduced) None

Hydrocortisone Warning

Levodopa Warning

Alpha-lipoic acid Warning

Nicotinamide Warning

Nicotinic acid Warning

Pyridoxine Warning

Pyruvic acid Warning

Thiamine None

L-tryptophan None

L-tyrosine Warning

Table 5A. Shows the compounds utilized in this project along with the GHS classification.

28

There are a number of classifications that indicate to the researcher the potential dangers of the

compound. The symbols are indicated with a red border around a rhombus with a small image in

the center. A health hazard indicates the possibility of a carcinogen, mutagenicity, reproductive

toxicity, respiratory sensitizer, target organ toxicity, and aspiration toxicity. This classification is

indicated by a person with a star. A gas cylinder indicates a gas under pressure and a flame over

a circle indicates the compound is an oxidizer. A flame shows the compound is flammable,

pyrophoric, self-heating, emits flammable gas, self-reactive, or is an organic peroxide. An image

of two test tubes reacting indicates the compound is classified as a corrosive; this can include

skin corrosion or burns, eye damage, or corrosive to metal. An environmental hazard can include

aquatic toxicity is indicated by an image of a dead fish and tree. An exclamation mark indicates

the compound is an irritant, skin sensitizer, an acute toxin, can cause narcotic effects, respiratory

tract issues, or is hazardous to the ozone layer. A compound is an explosive, self-reacting, or is

an organic peroxide if the symbol is an exploding bomb and a compound can cause acute toxicity

if the symbol is a skull and crossbones.15

29

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